Localisation of immune cell populations during human fetal testicular development.

graphical abstractABSTRACT: This study addresses the limited knowledge of immune cells and their origins in the human fetal testis. By identifying and localising macrophages, T cells, neutrophils, and mast cells, evidence of their potential roles in testicular and epididymal development is provided. Macrophages (CD68+), T cells (CD3+), neutrophils (CD66b+), and mast cells (tryptase+ and chymase+) were identified using immunohistochemistry and immunofluorescence in 20 formalin-fixed fetal testes (and epididymides, when present) collected at autopsy from stillborn fetuses (gestational weeks, GW14-41). Immune cell transcripts were compiled from published scRNA-seq datasets from human embryonic (GW6-8) and fetal (GW12-16) testes. CD68+ macrophages were abundant in interstitial, testis border, and perivascular regions, increasing adjacent to cords from GW23 onward. Interstitial CD3+ T cells were rare in second trimester but more frequent in the third. CD66b+ neutrophils were extremely rare. Chymase+ mast cells were absent, while tryptase+ mast cells were present near the capsule and vessels. scRNA-seq identified CD68-expressing cells as the predominant immune population from GW6-16. This first description of multiple immune cell types in the human fetal testis reveals similarities to murine testes; in both species, early macrophage abundance, preceding bone marrow haematopoiesis, suggests yolk sac and/or liver origins, while later-appearing immune cells are likely bone marrow-derived. The near-complete absence of Ly6G+ neutrophils in the later gestation stages differs from observations in mouse. These findings highlight potential developmental windows vulnerable to immune-related disruption and reinforce the value of mouse models for understanding human testis development. LAY SUMMARY: This study examines how the immune system helps the testicles to form before birth. In animals, immune cells called macrophages are important for early testis development, but their roles and those of other immune cells in humans were unclear. We studied tissues from 20 stillborn babies between 14 and 41 weeks of pregnancy to identify immune cells in developing testes and compared our results with existing genetic data. We found that macrophages were the most common immune cells, appearing early and increasing in number as the testis grew. Other immune cells, including T cells and mast cells, appeared later. Our findings suggest that macrophages come from early sources, such as the yolk sac or fetal liver, and play a key role in building the testis. Understanding these processes could reveal how factors affecting immune cell functions before birth could impair male fertility in men.